The lateral intercellular spaces (LIS) of MDCK cell epithelia grown on permeable supports are about 0.4 pH units acidic to the bathing solutions, presumably because of buffering by the fixed negative charges on the surface of the lateral cell membranes. To test the hypothesis that fixed buffers are responsible for the acidity, a theoretical and experimental approach was developed for the determination of the concentration and pK of the fixed buffer constituted by the glycocalyx. The pH of the solution in the LIS was measured by ratiometric fluorescence microscopy while the buffer concentration or composition of the bathing solutions was altered. In addition, the divalent cation Sr2+ was added to the perfusion solutions to displace protons from the fixed buffer sites for the determination of the fixed buffer properties. We conclude that the LIS contain 3.7 mm of pK 6.2 fixed buffer and that this buffer is responsible for the acidic microenvironment in the LIS.